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Gβγ subunits inhibit Epac-induced melanoma cell migration.

Baljinnyam E, Umemura M, De Lorenzo MS, Xie LH, Nowycky M, Iwatsubo M, Chen S, Goydos JS, Iwatsubo K - BMC Cancer (2011)

Bottom Line: We next examined the effect of mSIRK on Epac-induced Ca 2+ response.In addition, W-5, an inhibitor of calmodulin, abolished mSIRK's inhibitory effects on Epac-induced Ca 2+ elevation, and cell migration.These data suggest that, the mSIRK-induced Ca 2+ from the extracellular space inhibits the Epac-induced Ca 2+ release from the ER, resulting suppression of cell migration.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology and Molecular Medicine, New Jersey Medical School-University of Medicine and Dentistry of New Jersey, 185 South Orange Avenue, Newark 07103, USA.

ABSTRACT

Background: Recently we reported that activation of Epac1, an exchange protein activated by cAMP, increases melanoma cell migration via Ca 2+ release from the endoplasmic reticulum (ER). G-protein βγ subunits (Gβγ) are known to act as an independent signaling molecule upon activation of G-protein coupled receptor. However, the role of Gβγ in cell migration and Ca 2+ signaling in melanoma has not been well studied. Here we report that there is crosstalk of Ca 2+ signaling between Gβγ and Epac in melanoma, which plays a role in regulation of cell migration.

Methods: SK-Mel-2 cells, a human metastatic melanoma cell line, were mainly used in this study. Intracellular Ca 2+ was measured with Fluo-4AM fluorescent dyes. Cell migration was examined using the Boyden chambers.

Results: The effect of Gβγ on Epac-induced cell migration was first examined. Epac-induced cell migration was inhibited by mSIRK, a Gβγ -activating peptide, but not its inactive analog, L9A, in SK-Mel-2 cells. Guanosine 5', α-β-methylene triphosphate (Gp(CH2)pp), a constitutively active GTP analogue that activates Gβγ, also inhibited Epac-induced cell migration. In addition, co-overexpression of β1 and γ2, which is the major combination of Gβγ, inhibited Epac1-induced cell migration. By contrast, when the C-terminus of β adrenergic receptor kinase (βARK-CT), an endogenous inhibitor for Gβγ, was overexpressed, mSIRK's inhibitory effect on Epac-induced cell migration was negated, suggesting the specificity of mSIRK for Gβγ. We next examined the effect of mSIRK on Epac-induced Ca 2+ response. When cells were pretreated with mSIRK, but not with L9A, 8-(4-Methoxyphenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8-pMeOPT), an Epac-specific agonist, failed to increase Ca 2+ signal. Co-overexpression of β1 and γ2 subunits inhibited 8-pMeOPT-induced Ca 2+ elevation. Inhibition of Gβγ with βARK-CT or guanosine 5'-O-(2-thiodiphosphate) (GDPβS), a GDP analogue that inactivates Gβγ, restored 8-pMeOPT-induced Ca 2+ elevation even in the presence of mSIRK. These data suggested that Gβγ inhibits Epac-induced Ca 2+ elevation. Subsequently, the mechanism by which Gβγ inhibits Epac-induced Ca 2+ elevation was explored. mSIRK activates Ca 2+ influx from the extracellular space. In addition, W-5, an inhibitor of calmodulin, abolished mSIRK's inhibitory effects on Epac-induced Ca 2+ elevation, and cell migration. These data suggest that, the mSIRK-induced Ca 2+ from the extracellular space inhibits the Epac-induced Ca 2+ release from the ER, resulting suppression of cell migration.

Conclusion: We found the cross talk of Ca 2+ signaling between Gβγ and Epac, which plays a major role in melanoma cell migration.

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Gβγ inhibits Epac-induced cell migration. a) SK-Mel-2 cells were infected with adenovirus harboring LacZ or Epac1 followed by the migration assay in the presence of mSIRK or Gp(CH2)pp (10 μM). mSIRK inhibited Epac1-induced melanoma cell migration. *, p < 0.05 vs. LacZ control, #, p < 0.05 vs. Epac1 control. n = 4 except 5 and 10 μM of mSIRK (n = 2). b) Migration assay was performed in SK-Mel-2 cells in the presence or absence of 8-pMeOPT (200 μM), L9A (20 μM) or mSIRK (20 μM). mSIRK, but not L9A, inhibited 8-pMeOPT-induced cell migration. n = 4. c and d) Following the termination of adenovirus harboring LacZ or Epac1, SK-Mel-2 cells were subjected to co-overexpression of Gβ1 and Gγ2 subunits or βARK-CT. Western blot analyses showed increased expression of the target proteins. e) Migration assay was performed in SK-Mel-2 cells. Co-overexpression of Gβ1 and Gγ2 inhibited Epac1-induced cell migration. Overexpression of βARK-CT restored Epac1-induced cell migration even in the presence of mSIRK (20 μM). f) Ablation of Epac1 inhibits GPCR-induced cell migration in melanoma. SK-Mel-2 cells were infected with lentivirus harboring Epac1- or control-shRNA as we previously described [18]. Migration assay was performed in the presence or absence of isoproterenol (ISO) (100 μM), a β-adrenergic receptor agonist. Ablation of Epac1 inhibits both basal and ISO-induced cell migration. n = 4.
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Figure 1: Gβγ inhibits Epac-induced cell migration. a) SK-Mel-2 cells were infected with adenovirus harboring LacZ or Epac1 followed by the migration assay in the presence of mSIRK or Gp(CH2)pp (10 μM). mSIRK inhibited Epac1-induced melanoma cell migration. *, p < 0.05 vs. LacZ control, #, p < 0.05 vs. Epac1 control. n = 4 except 5 and 10 μM of mSIRK (n = 2). b) Migration assay was performed in SK-Mel-2 cells in the presence or absence of 8-pMeOPT (200 μM), L9A (20 μM) or mSIRK (20 μM). mSIRK, but not L9A, inhibited 8-pMeOPT-induced cell migration. n = 4. c and d) Following the termination of adenovirus harboring LacZ or Epac1, SK-Mel-2 cells were subjected to co-overexpression of Gβ1 and Gγ2 subunits or βARK-CT. Western blot analyses showed increased expression of the target proteins. e) Migration assay was performed in SK-Mel-2 cells. Co-overexpression of Gβ1 and Gγ2 inhibited Epac1-induced cell migration. Overexpression of βARK-CT restored Epac1-induced cell migration even in the presence of mSIRK (20 μM). f) Ablation of Epac1 inhibits GPCR-induced cell migration in melanoma. SK-Mel-2 cells were infected with lentivirus harboring Epac1- or control-shRNA as we previously described [18]. Migration assay was performed in the presence or absence of isoproterenol (ISO) (100 μM), a β-adrenergic receptor agonist. Ablation of Epac1 inhibits both basal and ISO-induced cell migration. n = 4.

Mentions: We have previously demonstrated that Epac increases melanoma cell migration by modification of heparan sulfate [17]. In addition, since it has been reported that Gβγ plays a role in cell migration of endothelial cells and breast cancer cells [4-6], we examined the effects of Gβγ on melanoma cell migration. We found that mSIRK, a cell-membrane permeable activator of Gβγ [7], decreased basal cell migration only at the highest dose (50 μM). In contrast, mSIRK inhibited Epac1 overexpression-induced cell migration in a dose-dependent manner (Figure 1a). These data suggest that the inhibitory effect of Gβγ is obvious under Epac-activated conditions, but not clear under basal conditions (Figure 1a). Gp(CH)2pp, a constitutively active GTP analogue which dissociates Gβγ from Gα, also inhibited Epac-induced cell migration. In addition, mSIRK inhibited cell migration induced by 8-pMeOPT, an Epac-specific agonist, suggesting that Gβγ also inhibits cell migration induced by endogenous Epac (Figure 1b). Further, we examined the specificity of Gβγ in the inhibition of Epac-induced cell migration. Co-overexpression of Gβ1 and Gγ2 subunits (Figure 1c), which is the major combination of Gβγ [25], inhibited Epac1-induced cell migration (Figure 1e). By contrast, overexpression of βARK-CT (Figure 1d), an inhibiting-peptide for Gβγ [26], abolished the mSIRK's inhibitory effect (Figure 1e). These data suggest that there is cross talk between Gβγ and Epac, which affects melanoma cell migration. Since activation of Epac was achieved by artificial overexpression or the Epac-agonist which does not exist in nature, we tested whether hormonal control of GPCR increases cell migration via Epac. Stimulation of β-adrenergic receptor increased cell migration in melanoma, and it was inhibited by ablation of Epac1 (Figure 1f), suggesting that Epac increases cell migration upon activation of hormone receptors.


Gβγ subunits inhibit Epac-induced melanoma cell migration.

Baljinnyam E, Umemura M, De Lorenzo MS, Xie LH, Nowycky M, Iwatsubo M, Chen S, Goydos JS, Iwatsubo K - BMC Cancer (2011)

Gβγ inhibits Epac-induced cell migration. a) SK-Mel-2 cells were infected with adenovirus harboring LacZ or Epac1 followed by the migration assay in the presence of mSIRK or Gp(CH2)pp (10 μM). mSIRK inhibited Epac1-induced melanoma cell migration. *, p < 0.05 vs. LacZ control, #, p < 0.05 vs. Epac1 control. n = 4 except 5 and 10 μM of mSIRK (n = 2). b) Migration assay was performed in SK-Mel-2 cells in the presence or absence of 8-pMeOPT (200 μM), L9A (20 μM) or mSIRK (20 μM). mSIRK, but not L9A, inhibited 8-pMeOPT-induced cell migration. n = 4. c and d) Following the termination of adenovirus harboring LacZ or Epac1, SK-Mel-2 cells were subjected to co-overexpression of Gβ1 and Gγ2 subunits or βARK-CT. Western blot analyses showed increased expression of the target proteins. e) Migration assay was performed in SK-Mel-2 cells. Co-overexpression of Gβ1 and Gγ2 inhibited Epac1-induced cell migration. Overexpression of βARK-CT restored Epac1-induced cell migration even in the presence of mSIRK (20 μM). f) Ablation of Epac1 inhibits GPCR-induced cell migration in melanoma. SK-Mel-2 cells were infected with lentivirus harboring Epac1- or control-shRNA as we previously described [18]. Migration assay was performed in the presence or absence of isoproterenol (ISO) (100 μM), a β-adrenergic receptor agonist. Ablation of Epac1 inhibits both basal and ISO-induced cell migration. n = 4.
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Figure 1: Gβγ inhibits Epac-induced cell migration. a) SK-Mel-2 cells were infected with adenovirus harboring LacZ or Epac1 followed by the migration assay in the presence of mSIRK or Gp(CH2)pp (10 μM). mSIRK inhibited Epac1-induced melanoma cell migration. *, p < 0.05 vs. LacZ control, #, p < 0.05 vs. Epac1 control. n = 4 except 5 and 10 μM of mSIRK (n = 2). b) Migration assay was performed in SK-Mel-2 cells in the presence or absence of 8-pMeOPT (200 μM), L9A (20 μM) or mSIRK (20 μM). mSIRK, but not L9A, inhibited 8-pMeOPT-induced cell migration. n = 4. c and d) Following the termination of adenovirus harboring LacZ or Epac1, SK-Mel-2 cells were subjected to co-overexpression of Gβ1 and Gγ2 subunits or βARK-CT. Western blot analyses showed increased expression of the target proteins. e) Migration assay was performed in SK-Mel-2 cells. Co-overexpression of Gβ1 and Gγ2 inhibited Epac1-induced cell migration. Overexpression of βARK-CT restored Epac1-induced cell migration even in the presence of mSIRK (20 μM). f) Ablation of Epac1 inhibits GPCR-induced cell migration in melanoma. SK-Mel-2 cells were infected with lentivirus harboring Epac1- or control-shRNA as we previously described [18]. Migration assay was performed in the presence or absence of isoproterenol (ISO) (100 μM), a β-adrenergic receptor agonist. Ablation of Epac1 inhibits both basal and ISO-induced cell migration. n = 4.
Mentions: We have previously demonstrated that Epac increases melanoma cell migration by modification of heparan sulfate [17]. In addition, since it has been reported that Gβγ plays a role in cell migration of endothelial cells and breast cancer cells [4-6], we examined the effects of Gβγ on melanoma cell migration. We found that mSIRK, a cell-membrane permeable activator of Gβγ [7], decreased basal cell migration only at the highest dose (50 μM). In contrast, mSIRK inhibited Epac1 overexpression-induced cell migration in a dose-dependent manner (Figure 1a). These data suggest that the inhibitory effect of Gβγ is obvious under Epac-activated conditions, but not clear under basal conditions (Figure 1a). Gp(CH)2pp, a constitutively active GTP analogue which dissociates Gβγ from Gα, also inhibited Epac-induced cell migration. In addition, mSIRK inhibited cell migration induced by 8-pMeOPT, an Epac-specific agonist, suggesting that Gβγ also inhibits cell migration induced by endogenous Epac (Figure 1b). Further, we examined the specificity of Gβγ in the inhibition of Epac-induced cell migration. Co-overexpression of Gβ1 and Gγ2 subunits (Figure 1c), which is the major combination of Gβγ [25], inhibited Epac1-induced cell migration (Figure 1e). By contrast, overexpression of βARK-CT (Figure 1d), an inhibiting-peptide for Gβγ [26], abolished the mSIRK's inhibitory effect (Figure 1e). These data suggest that there is cross talk between Gβγ and Epac, which affects melanoma cell migration. Since activation of Epac was achieved by artificial overexpression or the Epac-agonist which does not exist in nature, we tested whether hormonal control of GPCR increases cell migration via Epac. Stimulation of β-adrenergic receptor increased cell migration in melanoma, and it was inhibited by ablation of Epac1 (Figure 1f), suggesting that Epac increases cell migration upon activation of hormone receptors.

Bottom Line: We next examined the effect of mSIRK on Epac-induced Ca 2+ response.In addition, W-5, an inhibitor of calmodulin, abolished mSIRK's inhibitory effects on Epac-induced Ca 2+ elevation, and cell migration.These data suggest that, the mSIRK-induced Ca 2+ from the extracellular space inhibits the Epac-induced Ca 2+ release from the ER, resulting suppression of cell migration.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology and Molecular Medicine, New Jersey Medical School-University of Medicine and Dentistry of New Jersey, 185 South Orange Avenue, Newark 07103, USA.

ABSTRACT

Background: Recently we reported that activation of Epac1, an exchange protein activated by cAMP, increases melanoma cell migration via Ca 2+ release from the endoplasmic reticulum (ER). G-protein βγ subunits (Gβγ) are known to act as an independent signaling molecule upon activation of G-protein coupled receptor. However, the role of Gβγ in cell migration and Ca 2+ signaling in melanoma has not been well studied. Here we report that there is crosstalk of Ca 2+ signaling between Gβγ and Epac in melanoma, which plays a role in regulation of cell migration.

Methods: SK-Mel-2 cells, a human metastatic melanoma cell line, were mainly used in this study. Intracellular Ca 2+ was measured with Fluo-4AM fluorescent dyes. Cell migration was examined using the Boyden chambers.

Results: The effect of Gβγ on Epac-induced cell migration was first examined. Epac-induced cell migration was inhibited by mSIRK, a Gβγ -activating peptide, but not its inactive analog, L9A, in SK-Mel-2 cells. Guanosine 5', α-β-methylene triphosphate (Gp(CH2)pp), a constitutively active GTP analogue that activates Gβγ, also inhibited Epac-induced cell migration. In addition, co-overexpression of β1 and γ2, which is the major combination of Gβγ, inhibited Epac1-induced cell migration. By contrast, when the C-terminus of β adrenergic receptor kinase (βARK-CT), an endogenous inhibitor for Gβγ, was overexpressed, mSIRK's inhibitory effect on Epac-induced cell migration was negated, suggesting the specificity of mSIRK for Gβγ. We next examined the effect of mSIRK on Epac-induced Ca 2+ response. When cells were pretreated with mSIRK, but not with L9A, 8-(4-Methoxyphenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8-pMeOPT), an Epac-specific agonist, failed to increase Ca 2+ signal. Co-overexpression of β1 and γ2 subunits inhibited 8-pMeOPT-induced Ca 2+ elevation. Inhibition of Gβγ with βARK-CT or guanosine 5'-O-(2-thiodiphosphate) (GDPβS), a GDP analogue that inactivates Gβγ, restored 8-pMeOPT-induced Ca 2+ elevation even in the presence of mSIRK. These data suggested that Gβγ inhibits Epac-induced Ca 2+ elevation. Subsequently, the mechanism by which Gβγ inhibits Epac-induced Ca 2+ elevation was explored. mSIRK activates Ca 2+ influx from the extracellular space. In addition, W-5, an inhibitor of calmodulin, abolished mSIRK's inhibitory effects on Epac-induced Ca 2+ elevation, and cell migration. These data suggest that, the mSIRK-induced Ca 2+ from the extracellular space inhibits the Epac-induced Ca 2+ release from the ER, resulting suppression of cell migration.

Conclusion: We found the cross talk of Ca 2+ signaling between Gβγ and Epac, which plays a major role in melanoma cell migration.

Show MeSH
Related in: MedlinePlus